Conventional fluorescence lifetime imaging (“FLI”) suffers from at least two technical problems.
First, conventional FLI may require precise knowledge of the shape of the light waveform emitted by the FLI system to illuminate the scene. It is challenging to obtain a precise knowledge of the shape of the light waveform emitted by the FLI system, because physical processes (e.g., crosstalk, latency, electromagnetic interference, hysteresis, nonlinear responses, or signal noise) in electronics or other circuitry (e.g., in a computer, driver, light source, or wiring) may distort the shape (e.g., phase or amplitude) of the emitted light signal, relative to the shape of the electric control signal that is generated by a timer to control the lighting. For example, the electric control signal generated to control the lighting may be a square wave, but the actual emitted light waveform may be smoothed and curved. Such smoothing occurs in most electro-optical systems that tend to be low-pass systems that suppress high frequency components. In a conventional ToF system, in order to achieve precise knowledge of the shape of the light waveform that is emitted by the system, expensive equipment may be employed. This expensive equipment may be limited to a first mode (e.g., pulsed illumination with non-periodic pulses) or limited to a second mode (e.g., periodically modulated illumination, such as sinusoidal or square wave modulation), and thus (2) may be unable to function in both modes. As a result, conventional FLI cannot be performed by a general purpose, inexpensive consumer grade time-of-flight (“ToF”) camera.
Second, conventional FLI may require taking one or more calibration measurements to determine the placement of the fluorescent sample relative to the sensor. The distance or depth of the sample with respect to the sensor is an unknown that is typically calibrated. Typically, this calibration is repeated often, since a separate calibration is performed for each different scene being imaged. This repetitive, time-consuming calibration is not desirable.